Currently this project focuses on four key areas: (1) chemical synthesis of lead molecules and series identified by high-throughput screening against whole Mycobacterium tuberculosis (MTb), (2) the synthesis and evaluation of inhibitors of fumarase from tuberculosis, (3) the synthesis and evaluation of inhibitors of the inosine 5'-monophosphate dehydrogenase and (4) structure-based design and screening of inhibitors of NAD synthetase. Most of our effort currently is devoted to Project (1) in which we are screening large compounds libraries obtained from global collaborators including several large pharmaceutical companies to identify inhibitors of MTb growth under in vivo relevant conditions, performing dose-titration follow-up of hits and synthesizing or purchasing chemically similar compounds. These series are evaluated using secondary screens with a battery of conditions that are thought to be relevant during in vivo growth of MTb. We selected carbon source as an in vivo relevant variable since numerous studies have shown the metabolism of glucose, cholesterol and/ or other lipids are critical for growth and survival of this pathogen in host tissues. Since September 2014 we have completed hit confirmation screens by dose titration screens of the primary hits from a 270,000 compound deck from Merck under restricted growth conditions including a beta-oxidation substrate at low pH and nitrite, a 200,000 compound deck from AstraZeneca under 4 different growth conditions, a 40,000 compound deck from DuPont under two growth conditions and a 100,000 natural product collection from EISAI under 3 different growth conditions. Reconfirmed hits with IC50 values less than 10uM are then assayed for their ability to completely inhibit growth of the organism under a panel of in vitro growth conditions reflecting all carbon sources thought to be in vivo relevant by determination of the minimum inhibitory concentration (MIC) and parallel determination of their cytotoxicity to HepG2 cells. Every attempt is made to progress as many chemo-types as possible to increase the likelihood of hitting a diversity of targets. Hit series with multiple members showing activity for the scaffold with low-complexity, acceptable solubility and promising physicochemical properties for profiling are prioritized for follow-up to determine if the desirable balance of potency and ADME (absorption, distribution, metabolism and excretion) properties could be achieved in Lead Optimization. In contrast, series with structural alerts suggesting toxicophores are deprioritized. To rapidly expand the SAR for the prioritized chemotypes, commercially available analogs are purchased and tested in MIC assays. Target identification for prioritized series is initiated by mutation frequency analysis, whole genome resequencing of resistant isolates, microarray and metabolomics analyses. In addition, kinetic and thermodynamic solubility determinations and microsomal stability assays are done to further develop the information that will be essential to facilitate go / no-go progression into lead optimization. To date, we have identified hundreds of active series that require follow-up evaluation. Amongst these we have found many series of compounds that block the function of MmpL3, the trehalose monomycolate transporter essential for cell wall synthesis as well as several scaffolds that target the DprE1 epimerase involved in cell wall arabinan biosynthesis. Several kinase scaffolds were identified in the screens and SAR studies on these have suggested independent targets for a few of these whereas at least five scaffolds suggested the same target based on convergent SAR. Transcriptional profiling analyses indicated that at least 5 series affected cellular respiration. This was subsequently corroborated in mutant generation studies which demonstrated that mutations in qcrB, a component of the respiratory bc1 complex involved in aerobic respiration, conferred high-level resistance to these scaffolds. A mutant in an alternative oxygen-dependent respiratory complex was found to be hyper-susceptible to QcrB inhibitors and has proved to be a valuable strain to identify additional classes of bc1 complex inhibitors. We have determined that the majority of series that progressed into hit-to-lead chemistry, were discontinued due to failing biological target validation and have expended considerable effort in attempting to optimize the process of biological triage. Target identification for compounds with potent anti-tubercular activity has shown that certain pathways are apparently susceptible to a diversity of chemical scaffolds. These apparently promiscuous targets include several enzymes involved in mycobacterial cell wall mycolyl-arabinogalactan biosynthesis as well as the respiratory bc1 complex. Neither of these targets are thought to have significant promise for treating TB since cell wall targets are already well hit by existing TB drugs and show little promise for radically changing the duration of therapy. TB bacteria are largely only slowly replicating in human infections and appear relatively insensitive to inhibitors of cell wall enzymes. The development of additional drugs which target aspects of cell wall biosynthesis would thus be expected to have little effect on the duration of chemotherapy and would only be useful as new drugs to treat drug-resistant disease. We have found that inhibitors of the respiratory bc1 complex show strain dependence as well as medium dependence in their ability to inhibit growth. Metabolic and transcriptional analyses of MTb growing under hypoxia as well as of MTb recovered from rabbit and monkey granulomas, have indicated major alterations in utilization of components of respiratory pathways. These results suggest that careful target validation is essential before progressing any inhibitors of respiration and coupled energy generation into lead optimization. To optimize our chances of identifying series with the potential to improve the speed of cure of TB, we are now applying a 4-tiered approach to accelerating the process of drug development through early application of counter-screens and other informative assays that will increase early lead candidate attrition rates while decreasing attrition rates in the expensive later stages of drug development. In the first tier, validated hits from screens are assayed for potency against MTb under growth conditions similar to those encountered during infection, as well as their activity in reporter assays that measure cytotoxicity, mitochondrial toxicity, activity on promiscuous targets and ability to cause DNA damage. Compounds that are non-specifically cytotoxic or target promiscuous processes are de-prioritized. In the second tier, further deconvolution of mechanism of action takes place with a battery of assays that report on potential known mechanisms of action but that also report on detailed aspects of drug-target interaction through macromolecular incorporation assays, transcriptional profiling, as well as assessing their ability to cause cell lysis. In the third tier, we engage in-depth analyses for target identification or understanding mechanism of action on compounds by resistant mutant generation, analyses of kinetics of kill under replicating and non-replicating conditions, intra-macrophage activity and ensuring that the compounds retain potency against a panel of strains representing all major global strain lineages. In the fourth tier, target pathways are further validated by generation of regulated strains of genes in the pathway that can be tested in the right in vitro or in vivo disease model. In Projects 2-4 we are taking a variety of approaches to develop inhibitors and potential lead series against specific enzyme targets that are considered well validated as druggable in TB.